Renal clear-cell carcinoma (RCC) is the most prevalent and malignant histological type of kidney cancer, with no effective methods of treatment for metastatic disease. It is characterized by an early loss of the von Hippel- Lindau tumor-suppressor gene (VHL) in a large majority (60%-80%) of tumors. MicroRNAs (miRs) are short noncoding RNAs that bind to specific elements on mRNAs to repress translation of target proteins with regulatory functions in cancer. We have discovered that expression of mir204 is universally decreased in human RCC tumors, as compared with normal kidneys, and that the degree of reduced expression strongly correlates with tumor grade and cancer progression. Consistent with this, we found that miR204 has tumor- suppressing activity; it is cytotoxic to VHL(-) RCC cells in vitro and inhibits growth of tumors formed by these cells in nude mice. VHL positively regulates expression of miR204, and protects non-tumorigenic cells from cytotoxic activity of miR204. We further established that miR204 inhibits macroautophagy. Consistent with this, we found that MAP1LC3B (LC3B) protein, a regulator of macroautophagy, is a direct mir204 target. We also identified several other mir204 targets with potential roles in the regulation of macroautophagy. Macroautophagy is a specific program by which cells break down intracellular organelles to eliminate them, and also to obtain nutrients. Thus, macroautophagy serves as an internal source of nutrients to support the survival of cancer cells. Our general hypothesis is that loss of VHL during RCC promotes loss of mir204 and derepression of specific targets, thus activating LC3B-dependent macroautophagy, which gives cancer cells access to intracellular nutrients that contribute to their survival and to tumor growth. This renders RCC VHL(-) cells addicted to macroautophagic activity, and thus subject to synthetic lethality when macroautophagy is inhibited by exogenous mir204, with cell death caused by starvation. In contrast, the presence of VHL stimulates expression of endogenous mir204 and leads to suppression of the macroautophagic program, rendering the cells insensitive to the activity of exogenous mir204. To test this hypothesis, we will define the roles of mir204 and LC3B-dependent macroautophagy in RCC tumor formation (Aim 1), define the roles of other specific mir204 targets in macroautophagy and tumor formation (Aim 2); identify the molecular mechanisms leading to the decrease in mir204 expression in RCC (Aim 3); and determine levels of mir204 targets and general autophagic regulators in human RCC tumor and normal kidney samples (Aim 4). Impact: Here we provide evidence for a previously unknown signaling pathway activated by the losses of VHL and miR204 in RCC, which regulates survival of cancer cells. Understanding this pathway will lay the groundwork for the development of novel therapeutic approaches for the treatment of malignant RCC.